ACE2-binding exposes the SARS-CoV-2 fusion peptide to broadly neutralizing coronavirus antibodies

The coronavirus spike (S) glycoprotein attaches to host receptors and mediates viral fusion. Using a broad screening approach, we isolated from severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) immune donors seven monoclonal antibodies (mAbs) that bind to all human-infecting coronavirus S proteins. This class of mAbs recognize the fusion peptide and acquire affinity and breadth through somatic mutations. Despite targeting a conserved motif, only some mAbs show broad neutralizing activity in vitro against alpha- and beta-coronaviruses, including animal coronavirus WIV-1 and PDF-2180. Two selected mAbs also neutralize Omicron BA.1 and BA.2 authentic viruses and reduce viral burden and pathology in vivo. Structural and functional analyses show that the fusion peptide-specific mAbs bind with different modalities to a cryptic epitope, which is hidden in prefusion stabilized S, and becomes exposed upon binding of angiotensin-converting enzyme 2 (ACE2) or ACE2-mimicking mAbs.


BCR retrieval and molecular cloning
Clones with binding-profile-of interest after secondary screening were selected and their VH and VL sequences were obtained by reverse transcription PCR (RT-PCR) using Superscript III (Thermo Fisher, catalog no. 18080044) according to manufacturer's instruction, using the following VH, VK and VL-specific RT primers: HuIgG-const-anti 5'-TCTTGTCCACCTTGGTGTTGCT-3'; Hu-CK 5'-ACACTCTCCCCTGTTGAAGCTCTT-3' and Hu-CL 5'-ACTGTCTTCTCCACGGTGCT-3'. VH and VK/L sequences were amplified in two nested PCR reactions using adapted VH/VK/VL primers (75

Inference for unmutated common ancestor (UCA)
UCA inference were performed on both heavy and light chains for each mAb. Data were processed and analyzed using the Immcantation Framework (http://immcantation.org) with Change-O v1.0.2. First, the sequences were annotated using IgBlast version 1.16 (76) and IMGT as reference sequences (77). Second, clones were assigned based on IGHV genes, IGHJ gene, and junction distance with the Change-O DefineClones function. Germlines were then reconstructed using the Change-O CreateGermlines function. Finally, the phylogenic trees of each clone with their complete UCA sequences were generated with Igphyml (78).

Negative-stain electron microscopy
Three micromolar of the aforementioned purified SARS-CoV-2 S (D614G) ectodomain trimer were incubated with 3 µM of fusion peptide Fab for 1 h at room temperature prior the addition of 2.6 µM of S2X58 Fab. Incubation was continued at room temperature for 1 h after which samples were diluted to 0.01 mg/ml immediately before protein was adsorbed to glowdischarged manually carbon-coated copper grids for ~30 s before 2% uranyl formate staining.
Micrographs were recorded using the Leginon software (79) on a 120kV FEI Tecnai G2 Spirit with a Gatan Ultrascan 4000 4k × 4k CCD camera at 67,000 nominal magnification. The defocus ranged from 2.0 to 4.0 μm and the pixel size was 1.6 Å.

Epitope substitution scan
Epitope substitution scan was performed by PEPperPRINT GmbH, Heidelberg, Germany.
Briefly, each amino acid in the fusion peptide sequence K811PSKRSFIEDLLFNKVTLAD830 was substituted stepwise with all 20 main amino acids. These peptide variants and wildtype peptide, as well as HA control peptides was printed on a microarray chip in triplicate. Primary antibodies at 1 µg/ml (C13B8 and C13C9) or 100 µg/ml (VN01H1 and VP12E7) were incubated with the microarray chip for 16 h at 4ºC with orbital shaking at 140 rpm. After washing, secondary antibody goat anti-human IgG (H+L) DyLight680 (0.2 µg/ml) was incubated for 45 min at room temperature before reading on Innopsys InnoScan 710-IR Microarray Scanner.
Buffer exchange was performed using HiPrep 26/10 Desalting column and final product was sterilized through a 0.22 μm filter.

SARS-CoV-2 infection model in hamsters
KU LEUVEN R&D has developed and validated a SARS-CoV-2 Syrian Golden hamster infection model (90,91). This model is suitable for the evaluation of the potential antiviral activity and selectivity of novel compounds/antibodies (92). The SARS-CoV-2 strain used in the model, Gamma P.1 (EPI_ISL_1091366; 2021-03-08), was recovered from a nasopharyngeal swab taken from a traveler returning to Belgium in March 2021 (93). The variant was subjected to sequencing on a MinION platform (Oxford Nanopore) directly from the nasopharyngeal swabs;

SARS-CoV-2 RT-qPCR
Hamster lung tissues were collected after sacrifice and were homogenized using bead disruption (Precellys) in 350 µL TRK lysis buffer (E.Z.N.A. ® Total RNA Kit, Omega Bio-tek) and centrifuged (11,000 g, 5 min) to pellet cell debris. RNA was extracted according to the manufacturer's instructions. Of 50 μl eluate, 4 μl was used as a template in RT-qPCR reactions. RT-qPCR was performed on a LightCycler96 platform (Roche) using the iTaq Universal Probes One-Step RT-qPCR kit (BioRad) with N2 primers and probes targeting the nucleocapsid (90). Standards of SARS-CoV-2 cDNA (IDT) were used to express viral genome copies per mg tissue or per ml serum.

End-point virus titrations
Lung tissues were homogenized using bead disruption (Precellys) in 350 µl minimal essential medium and centrifuged (11,000 g, 5min, 4ºC) to pellet the cell debris. To quantify infectious SARS-CoV-2 particles, endpoint titrations were performed on confluent Vero E6 cells in 96well plates. Viral titers were calculated by the Reed and Muench method (96) using the Lindenbach calculator and were expressed as 50% tissue culture infectious dose (TCID 50 ) per mg tissue.

Histology
For histological examination, the lungs were fixed overnight in 4% formaldehyde and embedded in paraffin. Tissue sections (5 μm) were analyzed after staining with hematoxylin and eosin and scored blindly for lung damage by an expert pathologist. The scored parameters, to which a cumulative score of 1 to 3 was attributed, were the following: congestion, intra-alveolar hemorrhagic, apoptotic bodies in bronchus wall, necrotizing bronchiolitis, perivascular edema, bronchopneumonia, perivascular inflammation, peribranchial inflammation and vasculitis.

Models building and analysis
UCSF Chimera (97) and Coot (86) were used to align the crystal structures of Fabs-peptide complexes into the cryo-EM model PDB 6VXX. Model validation was done using Molprobity (98) and Phenix-Refine (87). Figures were generated using UCSF ChimeraX (99).

Fig. S1. An improved method that combines high-throughput screening and limiting dilution cloning to isolate B cells of unique characteristics.
Total PBMCs isolated from Ficoll density centrifugation were plated in replicate cultures at densities of 10 4 cells per well in the presence of 2.5 µg/ml of TLR agonist R848 and 1,000 U/ml IL-2. Six days later, the specificities of the secreted IgG antibodies in the culture supernatants from each culture were screened against different hCoVs S antigens in parallel (primary screening). Cultures that exhibit cross-reactive binding patterns (shown as red well) were next isolated as CD19 + IgMand IgAto enrich for IgG-secreting memory B cell blasts (100) and cloned by limiting dilution at 0.7 cell/well. Two days post cloning, the culture supernatants of the clones underwent secondary screening with the same panel of antigens to validate their binding profiles observed during primary screening. Clones which exhibit the same binding profiles as during the primary screening were selected for BCR retrieval by reverse transcription followed by nested PCR reactions. Paired IGH and IGK/IGL were cloned into expression vector and transfected into Expi293 cells for recombinant antibody expression. Recombinantly expressed mAbs were tested for their binding specificities to the S antigens as validation.      Fusion peptide-specific Fabs were incubated for 1 h with native-like soluble prefusion SARS-    Table S3. X-ray crystallography data collection and refinement statistics -Data in parentheses are for the highest resolution shell -R merge = ∑(∑|I i − 〈I〉|/∑|I|), where the first ∑ is the sum over all reflections, and the second ∑ is the sum over all measurements of a given reflection, with I i being the ith measurement of the intensity of the reflection and 〈I〉 the average intensity of that reflection. -R work /R free = ∑(|F o | − 〈|F c |〉)/∑|F o |, where 〈|F c |〉 is the expectation of |F c | under the probability model used to define the likelihood function. The sum is overall reflections.    (Fig. 1, A and D